Armored IL15 or IL15 receptor adaptors enhance proliferation and anti-tumor activity of CD19-targeted CAR-NK cells

YU Mengyuan; LIU Xiuying; ZHANG Xiaotian; WANG Jiaying; LING Zhiming; WANG Jianxun

doi:10.16016/j.2097-0927.202507093

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Armored IL15 or IL15 receptor adaptors enhance proliferation and anti-tumor activity of CD19-targeted CAR-NK cells

Basic Medicine | 更新时间：2025-12-24

- Armored IL15 or IL15 receptor adaptors enhance proliferation and anti-tumor activity of CD19-targeted CAR-NK cells
- Journal of Army Medical University Vol. 47, Issue 23, Pages: 2903-2912(2025)
- 作者机构：
  
  北京中医药大学生命科学学院，北京
- 作者简介：
  
  WANG Jianxun，E-mail： Jianxun.Wang@bucm.edu.cn
- 基金信息：
- DOI：10.16016/j.2097-0927.202507093
  CLC： R392.2;R73-362;R733.1
- Received：28 July 2025，
  
  Revised：2025-11-17，
  
  Accepted：24 November 2025，
  
  Published：15 December 2025
- 稿件说明：
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于梦圆，刘秀盈，章笑甜，等.装甲IL15或IL15受体连接体增强靶向CD19的CAR-NK细胞增殖与抗肿瘤活性［J］.陆军军医大学学报，2025，47（23）：2903-2912.DOI：10.16016/j.2097-0927.202507093.

YU M Y，LIU X Y，ZHANG X T，et al.Armored IL15 or IL15 receptor adaptors enhance proliferation and anti-tumor activity of CD19-targeted CAR-NK cells［J］.J Army Med Univ，2025，47（23）：2903-2912.DOI：10.16016/j.2097-0927.202507093.
于梦圆，刘秀盈，章笑甜，等.装甲IL15或IL15受体连接体增强靶向CD19的CAR-NK细胞增殖与抗肿瘤活性［J］.陆军军医大学学报，2025，47（23）：2903-2912.DOI：10.16016/j.2097-0927.202507093. DOI：

YU M Y，LIU X Y，ZHANG X T，et al.Armored IL15 or IL15 receptor adaptors enhance proliferation and anti-tumor activity of CD19-targeted CAR-NK cells［J］.J Army Med Univ，2025，47（23）：2903-2912.DOI：10.16016/j.2097-0927.202507093. DOI：

摘要

目的

构建靶向CD19的装甲白细胞介素15（interleukin 15，IL15）或IL15受体连接体（receptor-linker-IL15，RLI）嵌合抗原受体NK细胞（chimeric antigen receptor NK cell，CAR-NK），并进行CAR-NK细胞体外增殖与抗肿瘤活性的初步验证。

方法

培养人恶性非霍奇金淋巴瘤患者的自然杀伤细胞（NK92和NK92MI），并用逆转录病毒载体颗粒制备靶向CD19且装甲IL15或RLI的CAR-NK细胞。采用ELISA法检测CAR-NK细胞IL15分泌水平、CFSE法检测CAR-NK细胞的增殖能力。培养人B淋巴白血病细胞Nalm6-GFP-Luc和过表达人类CD19蛋白的人结肠癌细胞hCD19-SW620-GFP-Luc，并检测其表面CD19靶抗原的表达水平。在评估CAR-NK抗肿瘤活性时，使用荧光素酶发光法检测4或12 h时CAR-NK细胞在不同效应细胞与靶细胞数量比例时的杀伤效率，并利用流式细胞术检测CAR-NK细胞肿瘤杀伤时表面CD107a表达，同时使用CBA法检测其细胞因子释放水平。

结果

成功构建了装甲IL15或RLI的NK92/NK92MI-CD19 CAR细胞，且装甲IL15或RLI的细胞组IL15分泌水平显著高于没有装甲的细胞（

0.01）。在无IL-2刺激的情况下，IL15或RLI能够增强CAR-NK增殖能力（

0.05）。Nalm6-GFP-Luc和hCD19-SW620-GFP-Luc细胞表面CD19高表达，阳性率分别为100.0%和99.7%，可以作为CD19 CAR的靶细胞。IL15或RLI能够增强

NK92/NK92MI细胞来源的CAR-NK细胞的杀伤能力（

0.05），以及CD107a的表达水平。装甲IL15或RLI的NK92-CD19 CAR细胞有更高的IL2、IL10、IL6、TNF-α、sFasL、IFN-γ、Granulysin释放水平（

0.05），而装甲IL15或RLI的NK92MI-CD19 CAR细胞有更高的IL2、IL10、IL6、TNF-α、sFas、IFN-γ、Granzyme A、Granzyme B、Perforin、Granulysin释放水平（

0.05）。

结论

装甲IL15或RLI的NK92/NK92MI-CD19 CAR细胞具有良好的抗肿瘤活性，IL15细胞因子与CAR-NK联用具有强大的临床抗肿瘤治疗应用潜力。

Abstract

Objective

To construct CD19-targeted chimeric antigen receptor natural killer (CAR-NK) cells armored with interleukin15 (IL15) or IL15 receptor-linker (RLI) and preliminarily validate their proliferative capacity and anti-tumor activity in vitro.

Methods

Natural killer cells (NK92 and NK92MI) from patients with human malignant non-Hodgkin lymphoma were cultured

and CD19-targeted CAR-NK cells armored with IL15 or RLI were prepared using retroviral vector particles. IL15 secretion was measured by ELISA

and proliferative capacity was assessed via CFSE dilution assays. Human B-lymphocytic leukemia cells (Nalm6-GFP-Luc) and human colon cancer cells overexpressing CD19 (hCD19-SW620-GFP-Luc) were cultured

with surface CD19 expression confirmed (>99% positivity for both). Anti-tumor activity was evaluated by measuring cytotoxicity at effector-to-target (E:T) ratios using luciferase-based assays (4/12 h)

detecting surface CD107a expression via flow cytometry

and quantifying cytokine release using CBA assays.

Results

NK92/NK92MI-CD19 CAR cells armored with IL15 or RLI were successfully generated. IL15 secretion was significantly higher in armored groups versus non-armored controls (

0.01). Without IL-2 stimulation

IL15/RLI enhanced CAR-NK proliferation (

0.05). Both armored designs significantly increased tumor-killing efficiency (

0.05) and CD107a degranulation. IL15/RLI-arm

ored NK92-CD19 CAR cells exhibited elevated release of IL2

IL10

IL6

TNF-α

sFas

IFN-γ

and Granulysin (

0.05)

while armored NK92MI-CD19 CAR cells showed additional increases in Granzyme A

Granzyme B

and Perforin (

0.05).

Conclusion

IL15/RLI-armored NK92/NK92MI-CD19 CAR cells demonstrate potent anti-tumor activity

supporting their combined clinical therapeutic potential for tumors.

关键词

Keywords

references

FANG F ， XIAO W H ， TIAN Z G . Challenges of NK cell-based immunotherapy in the new era ［J］. Front Med ， 2018 ， 12 （ 4 ）： 440 - 450 . DOI： 10.1007/s11684-018-0653-9 http://dx.doi.org/10.1007/s11684-018-0653-9 .

VIVIER E ， RAULET D H ， MORETTA A ， et al . Innate or adaptive immunity—the example of natural killer cells ［J］. Science ， 2011 ， 331 （ 6013 ）： 44 - 49 . DOI： 10.1126/science.1198687 http://dx.doi.org/10.1126/science.1198687 .

GLIMCHER L ， SHEN F W ， CANTOR H . Identification of a cell-surface antigen selectively expressed on the natural killer cell ［J］. J Exp Med ， 1977 ， 145 （ 1 ）： 1 - 9 . DOI： 10.1084/jem.145.1.1 http://dx.doi.org/10.1084/jem.145.1.1 .

CHEEVER M A . Twelve immunotherapy drugs that could cure cancers ［J］. Immunol Rev ， 2008 ， 222 ： 357 - 368 . DOI： 10.1111/j.1600-065X.2008.00604.x http://dx.doi.org/10.1111/j.1600-065X.2008.00604.x .

BUDAGIAN V ， BULANOVA E ， PAUS R ， et al . IL-15/IL-15 receptor biology： a guided tour through an expanding universe ［J］. Cytokine Growth Factor Rev ， 2006 ， 17 （ 4 ）： 259 - 280 . DOI： 10.1016/j.cytogfr.2006.05.001 http://dx.doi.org/10.1016/j.cytogfr.2006.05.001 .

DESBOIS M ， BÉAL C ， CHARRIER M ， et al . IL-15 superagonist RLI has potent immunostimulatory properties on NK cells： implications for antimetastatic treatment ［J］. J Immunother Cancer ， 2020 ， 8 （ 1 ）： e000632 . DOI： 10.1136/jitc-2020-000632 http://dx.doi.org/10.1136/jitc-2020-000632 .

ZHAO L J ， QIU C T ， CHEN H Y ， et al . Construction of stable packaging cell lines for large-scale industrial BaEV-enveloped retroviral vector production ［J］. Front Immunol ， 2025 ， 16 ： 1578660 . DOI： 10.3389/fimmu.2025.1578660 http://dx.doi.org/10.3389/fimmu.2025.1578660 .

冯义超，刘秀盈，刘静静，等 . 基于逆转录病毒载体构建稳定表达人CD19和荧光蛋白的SW620细胞株［J］. 现代生物医学进展， 2024 ， 24 （ 4 ）： 612 - 616 . DOI： 10.13241/j.cnki.pmb.2024.04.003 http://dx.doi.org/10.13241/j.cnki.pmb.2024.04.003 .

FENG Y C ， LIU X Y ， LIU J J ， et al . Construction of a SW620 cell line stably expressing human CD19 and fluorescent protein based on retroviral packaging technology ［J］. Prog Mod Biomed ， 2024 ， 24 （ 4 ）： 612 - 616 . DOI： 10.13241/j.cnki.pmb.2024.04.003 http://dx.doi.org/10.13241/j.cnki.pmb.2024.04.003 .

ABEL A M ， YANG C ， THAKAR M S ， et al . Natural killer cells： development， maturation， and clinical utilization ［J］. Front Immunol ， 2018 ， 9 ： 1869 . DOI： 10.3389/fimmu.2018.01869 http://dx.doi.org/10.3389/fimmu.2018.01869 .

CHAPOVAL A I ， FULLER J A ， KREMLEV S G ， et al . Combination chemotherapy and IL-15 administration induce permanent tumor regression in a mouse lung tumor model： NK and T cell-mediated effects antagonized by B cells ［J］. J Immunol ， 1998 ， 161 （ 12 ）： 6977 - 6984 .

WANG W ， ERBE A K ， HANK J A ， et al . NK cell-mediated antibody-dependent cellular cytotoxicity in cancer immunotherapy ［J］. Front Immunol ， 2015 ， 6 ： 368 . DOI： 10.3389/fimmu.2015.00368 http://dx.doi.org/10.3389/fimmu.2015.00368 .

GONG J H ， MAKI G ， KLINGEMANN H G . Characterization of a human cell line （NK-92） with phenotypical and functional characteristics of activated natural killer cells ［J］. Leukemia ， 1994 ， 8 （ 4 ）： 652 - 658 .

余卓营，李晓瑞，尚凤琴，等 . 靶向CD19和BCMA的CAR-NK92MI细胞的构建及抗肿瘤活性研究［J］. 现代肿瘤医学， 2022 ， 30 （ 5 ）： 785 - 791 . DOI： 10.3969/j.issn.1672-4992.2022.05.006 http://dx.doi.org/10.3969/j.issn.1672-4992.2022.05.006 .

YU Z Y ， LI X R ， SHANG F Q ， et al . Construction and antitumor activity of CAR-NK92MI cells targeting CD19 and BCMA ［J］. J Mod Oncol ， 2022 ， 30 （ 5 ）： 785 - 791 . DOI： 10.3969/j.issn.1672-4992.2022.05.006 http://dx.doi.org/10.3969/j.issn.1672-4992.2022.05.006 .

KLOESS S ， OBERSCHMIDT O ， DAHLKE J ， et al . Preclinical assessment of suitable natural killer cell sources for chimeric antigen receptor natural killer-based “off-the-shelf ” acute myeloid leukemia immunotherapies ［J］. Hum Gene Ther ， 2019 ， 30 （ 4 ）： 381 - 401 . DOI： 10.1089/hum.2018.247 http://dx.doi.org/10.1089/hum.2018.247 .

PASLEY S ， ZYLBERBERG C ， MATOSEVIC S . Natural killer-92 cells maintain cytotoxic activity after long-term cryopreservation in novel DMSO-free media ［J］. Immunol Lett ， 2017 ， 192 ： 35 - 41 . DOI： 10.1016/j.imlet.2017.09.012 http://dx.doi.org/10.1016/j.imlet.2017.09.012 .

TAM Y K ， MARTINSON J A ， DOLIGOSA K ， et al . Ex vivo expansion of the highly cytotoxic human natural killer-92 cell-line under current good manufacturing practice conditions for clinical adoptive cellular immunotherapy ［J］. Cytotherapy ， 2003 ， 5 （ 3 ）： 259 - 272 . DOI： 10.1080/14653240310001523 http://dx.doi.org/10.1080/14653240310001523 .

JACOBS S K ， WILSON D J ， MELIN G ， et al . Interleukin-2 and lymphokine activated killer （LAK） cells in the treatment of malignant glioma： clinical and experimental studies ［J］. Neurol Res ， 1986 ， 8 （ 2 ）： 81 - 87 . DOI： 10.1080/01616412.1986.11739735 http://dx.doi.org/10.1080/01616412.1986.11739735 .

KU C C ， MURAKAMI M ， SAKAMOTO A ， et al . Control of homeostasis of CD8 + memory T cells by opposing cytokines ［J］. Science ， 2000 ， 288 （ 5466 ）： 675 - 678 . DOI： 10.1126/science.288.5466.675 http://dx.doi.org/10.1126/science.288.5466.675 .

SINDACO P ， PANDEY H ， ISABELLE C ， et al . The role of interleukin-15 in the development and treatment of hematological malignancies ［J］. Front Immunol ， 2023 ， 14 ： 1141208 . DOI： 10.3389/fimmu.2023.1141208 http://dx.doi.org/10.3389/fimmu.2023.1141208 .

WANG X ， ZHAO X Y . Transcription factors associated with IL-15 cytokine signaling during NK cell development ［J］. Front Immunol ， 2021 ， 12 ： 610789 . DOI： 10.3389/fimmu.2021.610789 http://dx.doi.org/10.3389/fimmu.2021.610789 .

VAN DEN BERGH J J ， SMITS E M ， VERSTEVEN M ， et al . Characterization of interleukin-15-transpresenting dendritic cells for clinical use ［J］. J Immunol Res ， 2017 ， 2017 ： 1975902 . DOI： 10.1155/2017/1975902 http://dx.doi.org/10.1155/2017/1975902 .

NORMAN D G ， BARLOW P N ， BARON M ， et al . Three-dimensional structure of a complement control protein module in solution ［J］. J Mol Biol ， 1991 ， 219 （ 4 ）： 717 - 725 . DOI： 10.1016/0022-2836（91）90666-t http://dx.doi.org/10.1016/0022-2836（91）90666-t .

DUBOIS S ， MAGRANGEAS F ， LEHOURS P ， et al . Natural splicing of exon 2 of human interleukin-15 receptor α-chain mRNA results in a shortened form with a distinct pattern of expression ［J］. J Biol Chem ， 1999 ， 274 （ 38 ）： 26978 - 26984 . DOI： 10.1074/jbc.274.38.26978 http://dx.doi.org/10.1074/jbc.274.38.26978 .

WU Z ， XUE H H ， BERNARD J ， et al . The IL-15 receptor　α chain cytoplasmic domain is critical for normal IL-15 Ralpha function but is not required for trans-presentation ［J］. Blood ， 2008 ， 112 （ 12 ）： 4411 - 4419 . DOI： 10.1182/blood-2007-03-080697 http://dx.doi.org/10.1182/blood-2007-03-080697 .

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